1. Field of the Invention
The present invention relates to the use of immunological reagents specific for a human transmembrane efflux pump protein (P-glycoprotein) in a biochemical conformation adopted in the presence of certain cytotoxic, lipophilic drugs that are substrates for P-glycoprotein, in the presence of cellular ATP depleting agents, and by certain mutant embodiments of Pgp. The invention provides such immunological reagents for immunodiagnostic and therapeutic uses, for isolating lymphocytes and hematopoietic stem cells, and for anticancer drug development.
2. Background of the Invention
Many human cancers express intrinsically or develop spontaneously resistance to several classes of anticancer drugs, each with a different structure and different mechanism of action. This phenomenon, which can be mimicked in cultured mammalian cells selected for resistance to certain A plant alkaloids or antitumor antibiotics such as colchicine, vinblastine and doxorubicin (formerly known as Adriamycin), is generally referred to as multidrug resistance (xe2x80x9cMDRxe2x80x9d; see Roninson (ed)., 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for Multidrug Resistance in Cancer, Academic Press, N.Y., Chapter 11 for reviews). The MDR phenotype presents a major obstacle to successful cancer chemotherapy in human patients.
MDR frequently appears to result from decreased intracellular accumulation of drug as a consequence of increased drug efflux related to alterations at the cellular plasma membrane. When mutant cell lines having the MDR phenotype are isolated, they are found to express an ATP-dependent non-specific molecular xe2x80x9cpumpxe2x80x9d protein (generally known as P-glycoprotein) that is located in the plasma membrane and keeps the intracellular accumulation of an anti-cancer drug low enough to evoke the drug-resistance phenotype. This protein (which has been determined to be the gene product of the MDR1 gene in humans) facilitates active (i.e., energy-dependent) drug efflux from the cell, against a concentration gradient of (generally) lipophilic compounds, including many cytotoxic drugs.
The gene encoding P-glycoprotein (which is also known as gp170-180 and the multidrug transporter) has been cloned from cultured human cells by Roninson et al. (see co-owned U.S. Pat. No. 5,206,352, issued Apr. 27, 1993, having an effective filing date of Mar. 28, 1986), and is generally referred to as MDR 1. The protein product of the MDR 1 gene, most generally known as P-glycoprotein (xe2x80x9cPgpxe2x80x9d), is a 170-180 kilodalton (kDa) transmembrane protein having the aforementioned energy-dependent efflux pump activity.
Molecular analysis of the MDR1 gene indicates that Pgp consists of 1280 amino acids distributed between two homologous halves (having 43% sequence identity of amino acid residues), each half of the molecule comprising six hydrophobic transmembrane domains and an ATP binding site within a cytoplasmic loop. Only about 8% of the molecule is extracellular, and carbohydrate moieties (approximately 30 kDa) are bound to sites in this region (Chen et al., 1986, Cell 47: 381-387).
Expression of Pgp on the cell surface is sufficient to render cells resistant to many (but not all) cytotoxic drugs, including many anti-cancer agents. Pgp-mediated MDR appears to be an important clinical component of drug resistance in tumors of different types, and MDR1 gene expression correlates with resistance to chemotherapy in different types of cancer.
Because Pgp is involved in the resistance of different types of human malignancies to conventional chemotherapy, the expression of Pgp is an important diagnostic and prognostic factor which in many cases helps the physician to choose the most effective combination of chemotherapeutic drugs and to monitor the efficacy of treatment. One way Pgp expression has been evaluated is by detecting the binding of specific immunological reagents (antibodies) to tumor samples. However, frequently the expression level of Pgp in tumor cells is low and cannot be reproducibly detected by routine immunological methods. In addition, there are few immunological or other reagents specific for functionally-active Pgp (which are the only forms of Pgp that are clinically relevant). Thus, there is a need in the art to increase the sensitivity and specificity of immunological and immunohistochemical methods for detecting functional Pgp expression.
Pgp is also constitutively expressed in many normal cells and tissues (see Cordon-Cardo et al., 1990, J. Histochem. Cytochem. 3: 1277; and Thiebaut et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7735 for reviews). In hematopoietic cells, Neyfakh et al. (1989, Exp. Cancer Res. 185-496) have shown that certain subsets of human and murine lymphocytes efflux Rhl23, a fluorescent dye that is a Pgp substrate, and this process can be blocked by small molecule inhibitors of Pgp. It has been demonstrated more recently that Pgp is expressed on the cell-surface membranes of pluripotent stem cells, NK cells, CD4- and CD8-positive T lymphocytes, and B lymphocytes (Chaudhary et al., 1992, Blood 8: 2735; Drach et al., 1992, Blood 80: 2729; Kimecki et al., 1994, Blood 83: 2451; Chaudhary et al., 1991, Cell 66: 85). Pgp expression on the cell surface membranes of different subsets of human lymphocytes has been extensively documented (Coon et al., 1991, Human Immunol. 32: 134; Tiirikainen et al., 1992, Ann. Hematol. 65: 124; Schluesener et al., 1992, Immunopharmacology 23: 37; Gupta et al., 1993, J. Clin. Immunol. 13: 289). Although recent studies suggest that Pgp plays a role in normal physiological functions of immune cells (Witkowski et al., 1994, J. Immunol. 153: 658; Kobayashi et al., 1994, Biochem. Pharmacol. 48: 1641; Raghu et al., 1996, Exp. Hematol. 24: 1030-1036, as disclosed more fully in co-pending U.S. patent application Ser. No. 08/658,583, filed Jun. 7, 1996, incorporated by reference herein in its entirety), the physiological role of Pgp in normal immune cells has remained unclear to date.
Expression of Pgp in hematopoietic cells provides an effective means for identifying and purifying lymphocytes and hematopoietic stem cells. As described more completely in co-owned and/or co-pending U.S. Pat. No. 5,434,075, issued Jul. 18, 1995 and U.S. patent application Ser. No. 08/032,056, filed Mar. 16, 1993, functional Pgp assays (such as fluorescent dye efflux) and immunochemical methods (such as fluorescence activated cell sorting (FACS) analysis) can in theory be used to purify lymphocytes and hematopoietic stem cells.
However, the levels of expression of Pgp on stem cells are low, and consequently the amount of an immunological reagent such as a monoclonal antibody (mAb) bound to a hematopoietic stem cell membrane using conventional procedures is generally not high enough to efficiently separate Pgp-positive cells by any conventional immunological technique (such as FACS, immunomagnetic particle separation, cell panning, or other methods known in the art). Thus, there remains a need in this art to improve the efficiency of methods for using Pgp expression to specifically purify lymphocytes and hematopoietic stem cells from biological sources.
Once the central role in MDR played by Pgp was uncovered, agents with a potential for reversing MDR phenotypes were developed that target Pgp. Several classes of drugs, including calcium channel blockers (e.g., verapamil), immunosuppresants (such as cyclosporines and steroid hormones), calmodulin inhibitors, and other compounds, were found to enhance the intracellular accumulation and cytotoxic action of Pgp-transported drugs (Ford et al., 1990, Pharm. Rev. 42: 155). Many of these agents were found to inhibit either drug binding or drug transport by Pgp (Akiyama et al., 1988, Molec. Pharm. 3: 144; Horio et al., 1988, Proc. Natl. Acad. Sci. USA 84: 3580). Some of these agents themselves were found to bind to and be effluxed by Pgp, suggesting that their enhancing effects on the cytotoxicity of Pgp substrates are due, at least in part, to competition for drug binding sites on this protein (Cornwell et al., 1986, J. Bio. Chem. 261: 7921; Tamai, 1990, J. Biochem. Molec. Biol. 265: 16509).
Many of these agents, however, also have strong, deleterious side effects at physiologically-achievable concentrations. These systemic side effects severely limit the clinical use of these agents as specific inhibitors of Pgp or for negative selection against Pgp-expressing tumor cells. Most of the known MDR-reversing drugs used in clinical trials have major side effects unrelated to inhibition of Pgp, such as calcium channel blockage (verapamil) or immunosuppression (cyclosporines and steroids). Similarly, targeting of cytotoxic drugs to Pgp-expressing cells is capable of compromising normal tissue function in normal cells (such as kidney, liver, colonic epithelium, etc.) that normally express Pgp. These drawbacks restrict the clinically-achievable dose of such agents and ultimately, their usefulness.
Immunological reagents, specifically such agents linked to cytotoxic molecules or detectably labeled reporter molecules, provide an alternate and specific way for identifying cells expressing Pgp at the cell surface and specifically delivering cytotoxic substances directly to such cells. Immunological reagents specific for extracellular epitopes of Pgp, such as anti-Pgp antibodies, offered the prospect of specificity, since antibodies should target only Pgp. However, it has also been recognized that only antibodies which react with an extracellular epitope of Pgp are expected to react with the protein in the plasma membrane of intact cells and thereby inhibit the MDR phenotype in such cells. Antibodies directed to the cytoplasmic portion of Pgp, on the other hand, are unlikely to be useful for reversal of MDR.
In addition, antibody binding to Pgp was expected to have a more-prolonged inhibitory effect than that caused by transient binding of a competitive inhibitor. Such reagents may also provide a means for delivering cytotoxic agents specifically to Pgp-expressing tumor cells in regimens aimed to selective killing of such cells.
Monoclonal antibodies specific for Pgp are known in the art.
Hamada et al., 1986, Proc. Natl. Acad. Sci. USA 83: 7785 disclose the mAbs MRK-16 and MRK-17, produced by immunizing mice with doxorubicin-resistant K-562 human leukemia cells. MRK-16 mAb was also reported to modulate vincristine and actinomycin D transport in resistant cells, and MRK-17 was shown to specifically inhibit growth of resistant cells with these drugs. Meyers et al., 1987, Cancer Res. 49: 3209 disclose mAbs HYB-241 and HYB-612, which recognize an external epitope of Pgp.
O""Brien et al., 1989, Proc. Amer. Assoc. Cancer Res. 30:Abs 2114 disclose that mAbs HYB-241 and HYB-612 increased the accumulation of vincristine and actinomycin D in tumor cells and increased the cytotoxicity of combinations of these drugs with verapamil.
Tsuruo et al., 1989, Jpn. J. Cancer Res. 80: 627 reported that treatment of athymic mice that had been previously inoculated with drug resistant human ovarian cancer cells with the mAb MRK 16 caused regression of established subcutaneous tumors.
Hamada et al., 1990, Cancer Res. 50: 3167 disclosed a recombinant chimeric antibody that combines the variable region of MRK-16 with the Fc portion of a human antibody, and showed this chimeric antibody to be more effective than MRK-16 mAb in increasing cytotoxicity in vitro.
Pearson et al., 1991, J. Natl. Cancer Inst. 88: 1386 disclosed that MRK-16 mAb increased the in vivo toxicity of vincristine to a human MDR colon cancer cell line grown as a xenograft in nude mice. The in vitro potentiation of drug cytotoxicity by MRK-16 mAb was, however, weak relative to known chemical inhibitors of Pgp action, and was apparently limited to only two Pgp substrates (vincristine and actinomycin D), having no effect on cytotoxicity by doxorubicin.
Cinciarelli et al., 1991, Int. J. Cancer 47: 533 disclosed a mouse IgG2, mAb, termed MAb657, having cross reactivity to Pgp-expressing human MDR cells. This mAb was shown to increase the susceptibility of MDR cells to human peripheral blood lymphocyte-mediated cytotoxicity, but was not shown to have an inhibitory effect on the drug efflux activity of Pgp.
Arcesi et al., 1993, Cancer Res. 53: 310-317 disclosed mAb 4E3 that binds to extracellular epitopes of Pgp but does not disrupt drug efflux or potentiate MDR drug-induced cytotoxicity.
Mechetner and Roninson, in co-owned and/or co-pending U.S. Pat. No. 5,434,075, issued Jul. 18, 1995, and in U.S. patent application Ser. No. 08/032,056, filed Mar. 16, 1993, disclosed mAb UIC2, having specificity for extracellular Pgp epitopes. This antibody was also shown to effectively inhibit Pgp-mediated drug efflux in MDR cells, and to reverse the MDR phenotype in vitro thereby, for a number of structurally and functional different cytotoxic compounds and all tested chemotherapeutic drugs known to be substrates for Pgp-mediated drug efflux.
The production of UIC2 mAb demonstrated the usefulness of the development of mAbs specific for extracellular epitopes of Pgp that were capable of inhibiting drug efflux activity. As evidenced by the mAbs developed in the prior art, production of extracellular epitope-specific mAbs does not necessarily result in mAbs that can affect drug efflux. There thus remains in the art a need for developing methods for producing mAbs that are capable of inhibiting drug efflux activity in Pgp. There also remains a need in the art for methods for developing more sensitive mAbs and methods to improve the sensitivity of currently available mAbs for the detection of Pgp expression in cancer cells in vivo, for improved cancer diagnostics and therapeutic applications with both normal and tumor cells expressing Pgp. There also remains a need in the art to develop more specific and efficient tools for the isolation of lymphocytes and hematopoietic stem cells, especially pluripotent and totipotent stem cells.
The present invention provides methods for production of mAbs specific for certain Pgp mutants and for Pgp in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents. The invention also provides methods for improving the sensitivity of and developing mAbs specific for Pgp in said biochemical conformation, and thereby provides improved cancer diagnostic and therapeutic methods and methods for developing anticancer drugs, and improved methods for blood stem cell purification. These methods are all based on the discovery by the present inventors that certain mAbs, particularly UIC2, are specific for Pgp in a biochemical conformation adopted in certain mutant embodiments of Pgp and in the presence of Pgp-mediated transport substrates and ATP depleting agents. The methods of the invention are based on enhanced antibody binding to Pgp in the presence of Pgp-mediated transport substrates or ATP depleting agents.
In a first aspect, the invention provides a method for producing an immunological reagent specific for P-glycoprotein in a biochemical conformation adopted by certain Pgp mutants and by Pgp in the presence of Pgp-mediated transport substrates or ATP depleting agents. In this aspect, the method comprises the steps of introducing a cell expressing a heterologous P-glycoprotein into an animal syngeneic with the species from which the cell was derived, wherein the heterologous Pgp molecule is in said biochemical conformation. The invention thus provides a method for producing immune cells in the animal expressing an antibody specific for this biochemical conformation of P-glycoprotein. In a preferred embodiment, the method of the invention provides a polyclonal antisera specific for P-glycoprotein in said biochemical conformation. In more preferred embodiments, the invention provides a monoclonal antisera specific for P-glycoprotein in said biochemical conformation. In the most preferred embodiment, the invention provides a hybridoma cell line that produces a monoclonal antibody specific for P-glycoprotein in said biochemical conformation. The invention also provides a monoclonal antibody produced using the methods of the invention.
In this aspect of the invention, a preferred embodiment of the P-glycoprotein in this specific biochemical conformation is achieved by providing a heterologous Pgp protein wherein particular amino acid residues in the ATP binding site of each half of the Pgp molecule are altered to provide a mutant or variant Pgp molecule. In preferred embodiments, the heterologous P-glycoprotein expressing-syngeneic cells express a mutant P-glycoprotein wherein each of the ATPase-specific active sites carry mutations that prevent ATP binding and/or ATP hydrolysis by these mutant Pgp proteins. In preferred embodiments, such mutants are characterized by amino acid substitution mutations in active site amino acid residues. In certain preferred embodiments, the substituted amino acid residues are lysine residues in the ATPase sites. In particularly preferred embodiments, the mutant the Pgp protein is human Pgp wherein the lysine residues at positions 433 and 1076 of the 1280 Pgp amino acid sequence are substituted with another amino acid, preferably methionine. In other preferred embodiments, the heterologous P-glycoprotein expressing-syngeneic cells express a mutant P-glycoprotein having amino acid substitution mutations at ATPase active site residues are glycine residues. In particularly preferred embodiments, the mutant the Pgp protein is human Pgp having glycine residues at positions 432 and 1075 of the 1280 Pgp amino acid sequence, preferably with serine residues.
In a second aspect, the invention provides a method for detecting functional P-glycoprotein expression in a mammalian cell, particularly a malignant mammalian cell and most particularly a multidrug resistant malignant mammalian cell. In this aspect of the invention the method comprises the steps of: (a) treating the mammalian cell with a P-glycoprotein substrate selected from the group consisting of reserpine, gramicidin, cyclosporine, vincristine, actinomycin D, taxol, verapamil and vinblastine or with an ATP-depleting agent; and then (b) reacting the mammalian cell with a detectably-labeled immunological reagent specific for P-glycoprotein in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents; and (c) detecting specific binding of the immunological reagent to the mammalian cell in the presence of the Pgp substrate or ATP depleting agent. In a preferred embodiment, the immunological reagent is a monoclonal antibody specific for P-glycoprotein in said biochemical conformation. In preferred embodiments, the immunological reagent is specific for a mutant form of Pgp wherein each of the lysine residues in the ATPase-specific active site of each half of the Pgp molecule has been changed to a residue other than lysine and preferably methionine. In a most preferred embodiment, the immunological reagent is the UIC2 monoclonal antibody (A.T.C.C. Accession No. HB 11027). Preferably, specific binding of the immunological reagent is increased in the presence of the Pgp substrate or ATP-depleting agent.
In a third aspect, the invention provides improved methods for functional P-glycoprotein specific staining using methods well-known in the art, including fluorescence-activated cell sorting, immunohistochemistry and similar staining methods. The invention also provides methods for discriminating between Pgp-specific and non-specific cell staining, whereby specific staining is associated with enhanced mAb staining of Pgp-expressing cells in the presence of a Pgp substrate or ATP depleting agent. In this aspect of the invention are provided methods wherein P-glycoprotein staining is achieved in the presence of a Pgp-mediated transport substrate or ATP-depleting agent, using an immunological reagent of the invention specific for Pgp in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents. In a preferred embodiment, the immunological reagent is a monoclonal antibody specific for P-glycoprotein in said biochemical conformation. In preferred embodiments, the immunological reagent is specific for a mutant form of Pgp wherein each of the lysine residues in the ATPase-specific active site of each half of the Pgp molecule has been changed to a residue other than lysine and preferably methionine. In a most preferred embodiment, the immunological reagent is the UIC2 monoclonal antibody (A.T.C.C. Accession No. HB 11027).
In a fourth aspect, the invention provides a method for identifying an immunological reagent, comprising antisera, antibodies, preferably monoclonal antibodies, and proteolytic or other Pgp-binding fragments thereof that are specific for P-glycoprotein in a biochemical conformation adopted by certain mutant embodiments of Pgp, and by Pgp in the presence of Pgp-mediated transport substrates or ATP depleting agents. This aspect of the methods of the invention is comprised of the steps: (a) reacting a mammalian cell expressing Pgp with a monoclonal antibody to be tested in the presence and absence of a P-glycoprotein substrate or ATP-depleting agent; and (b) detecting an increase in binding of a monoclonal antibody specific for P-glycoprotein in said biochemical conformation in the presence of a P-glycoprotein substrate or ATP-depleting agent. Preferably, the P-glycoprotein substrate is selected from the group consisting of reserpine, gramicidin, cyclosporine, vincristine, actinomycin D, taxol, verapamil and vinblastine.
In a fifth aspect, the invention provides a method for detecting and purifying lymphocytes and hematopoietic stem cells from a mammal, wherein the method comprising the steps of: (a) treating a biological sample comprising lymphocytes or hematopoietic stem cells with a P-glycoprotein substrate or ATP-depleting agent; (b) reacting the biological sample with a detectably-labeled immunological reagent specific for P-glycoprotein in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents; and (c) separating the lymphocytes or hematopoietic stem cells reacted with the detectably-labeled immunological reagent from the biological sample. In a preferred embodiment, the P-glycoprotein substrate is selected from the group consisting of non-toxic Pgp substrate, preferably cyclosporine and non-toxic derivatives thereof, and verapamil. In a preferred embodiment, the biological sample comprises blood, cord blood, lymph or bone marrow, with or without prior drug treatment. In a preferred embodiment, the immunological reagent is labeled with a detectable label, such as a fluorescent label, and the lymphocytes or hematopoietic stem cells reacted with the fluorescently-labeled immunological reagent. In preferred embodiments, lymphocytes or hematopoietic stem cells reacted with the detectably-labeled immunological reagent are separated from the biological sample by fluorescence-activated cell sorting, cell panning, immunomagnetic particles and other cell-separating means known in the art.
In a sixth aspect, the invention provides a method for improving detection of low levels of Pgp expression in mammalian cells, most preferably malignant mammalian cells and cells expressing the MDR phenotype, using the immunological detection methods of the invention. In preferred embodiments, the immunological detection methods include, but are not limited to, fluorescence activated cell sorting (FACS), most preferably providing an improvement of the sensitivity of FACS detection and isolation of cells expressing P-glycoprotein. In this aspect, the method comprises treating a population of mammalian cells comprising a mammalian cell expressing P-glycoprotein with a P-glycoprotein substrate or ATP depleting agent; reacting the population of mammalian cells with a fluorescently-labeled immunological reagent specific for P-glycoprotein in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents; and performing an immunological detection method such as fluorescence activated cell sorting on the mammalian cells. In a preferred embodiment, the P-glycoprotein substrate is selected from the group consisting of reserpine, gramicidin, cyclosporine, vincristine, actinomycin D, taxol, verapamil and vinblastine. In a preferred embodiment, the immunological reagent is a monoclonal antibody specific for P-glycoprotein in said biochemical conformation. In preferred embodiments, the immunological reagent is specific for a mutant form of Pgp wherein each of the lysine residues in the ATPase-specific active site of each half of the Pgp molecule has been changed to a residue other than lysine and preferably methionine. In a most preferred embodiment, the immunological reagent is the UIC2 monoclonal antibody (A.T.C.C. Accession No. HB 11027).
In a seventh aspect, the invention provides methods for identifying and selectively eliminating tumor cells expressing functional Pgp. In this aspect of the invention, the method comprises treatment of a mammalian cell, preferably a tumor cell, expressing functional P-glycoprotein with an immunological reagent, preferably a monoclonal antibody specific for Pgp in a biochemical conformation adopted in the presence of a Pgp substrate or ATP depleting agent, said treatment also being in the presence of a Pgp substrate or ATP depleting agent, whereby the immunological reagent further comprises a cytotoxic agent. In a preferred embodiment, the immunological reagent is a monoclonal antibody specific for P-glycoprotein in said biochemical conformation. In preferred embodiments, the immunological reagent is specific for a mutant form of Pgp wherein each of the lysine residues in the ATPase-specific active site of each half of the Pgp molecule has been changed to a residue other than lysine and preferably methionine. In a most preferred embodiment, the immunological reagent is the UIC2 monoclonal antibody (A.T.C.C. Accession No. HB 11027).
Also provided by the invention are methods for determining the antigenic epitope(s) of P-glycoprotein involved in mAb UIC2 binding and methods for producing antibodies specific for such epitopes.
The invention also provides methods for discriminating between multidrug resistance in mammalian cells resulting from the expression of functional Pgp and multidrug resistance related to the expression of the multidrug resistance related protein (the MRP gene product). In this aspect, the method comprises a showing of enhanced mAb binding in the presence of a Pgp substrate or ATP depleting agent as being specific for Pgp-mediated multidrug resistance.
Use of the methods of the invention for medical diagnostics of primary and malignant disease for detecting expression, particularly low-level expression, of P-glycoprotein is also provided by the invention.
The invention also provides a means for evaluating novel cytotoxic, chemotherapeutic drugs and Pgp inhibitors. The existing methods for screening and testing of new drugs that are Pgp inhibitors are based on the cytotoxicity or dye exclusion assays. These methods are costly, laborious and time-consuming. A screening test based on the enhanced binding of UIC2 mAb or its derivatives in the presence of Pgp substrates enables the rapid, reliable and cost-effective characterization of potential new Pgp-targeted drugs.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.